Outpatient Department before initiation of peripheral blood cell (PBC) collections. The 
last dose will be administered after the first collection of PBC. Injections must be 
scheduled to begin on a Friday , in order for leukapheresis and laboratory processing to 
be performed on consecutive weekdays of the following week. 
C. Peripheral blood cell collection 
PBC are collected by continuous flow centrifugation on days 4 (Monday) and 5 after the 
first dose of G-CSF with an expected yield of 5 - 10 x 10 8 /kg peripheral blood 
mononuclear cells. Collections will take place in the FHCRC Pheresis Unit under the 
supervision of Dr. Bensinger. Venous access will be accomplished using a temporary 
percutaneous Mahurkar catheter. Patients should receive a 7-day course of dicloxacillin 
or another appropriate antibiotic, starting on the day of line placement. 
D. Separation of CD34 + peripheral blood cells 
To simplify the transduction procedure by reducing the cell number manipulated and to 
probably increase the efficiency of gene transduction by improving the ratio of 
repopulating cell to vector particles, peripheral blood mononuclear cells will be enriched 
for CD34+ cells using the CellPro Stem Cell Separator. Mononuclear cells will be 
incubated with biotinylated murine antihuman CD34 antibody 12.8, washed and passed 
over an avidin biogel column. The cells adhering to the column will be dislodged by 
mechanical agitation and collected. A sample of CD34+ cells will be saved for FACS 
analysis and for use as pre-transduction control for PCR and for glucocerebrosidase 
enzyme assay. 
E. Transduction of CD34+ peripheral blood cells 
CD34+ peripheral blood cells will be transduced by incubation for 5 days in a long-term 
marrow culture system containing vector supernatant. Adherent stromal layers of LTMC 
will be established with autologous marrow cells 14 days before start of transduction. 
LTMC will contain 50% LTMC medium: Iscove’s modified Dulbecco’s Medium at 365 
mOsM supplemented with 12.5% fetal calf serum, 12.5% horse serum, 100 U/ml 
penicillin, 100 pg/ml streptomycin sulfate, 0.4 mg/ml of L-glutamine, 0.01 mg/ml folic 
acid, 0.04 mg/ml myoinositol, 0.01 mM 2-mercaptoethanol, and 1 |ig/ml hydrocortisone 
sodium succinate. LTMC will also contain rhIL-1, rhIL-3, rhIl-6, rhSCF (each at 50 
ng/ml), 50% vector-containing supernatant from PG13/LgGC cell cultures and 5 pg/ml 
protamine sulfate. LTMC will be fed every day with 50% fresh vector-containing 
supernatant and 50% used LTMC medium. After 5 days, adherent and nonadherent 
cells will be harvested from the LTMC and washed. Cell samples will be taken to test 
for viability using trypan exclusion and colony assays and for sterility using gram stain, 
culture for bacteria and fungi, mycoplasma screen and endotoxin test. Pooled 
supernatants from the transduction step will be analyzed for replication competent 
retrovirus using an S + L- focus assay on PG-4 cells. Cell samples will also be tested for 
transduction efficiency by PCR, Southern blot, and glucocerebrosidase enzyme assay. 
Transduced cells will only be used for infusion into the patient if gram stain is negative. 
F. Infusion of transduced CD34+ peripheral blood cells 
CD34+ peripheral blood cells will be infused into the patient over a period of two hours 
or less. Patients will receive premedication 30 minutes before start of infusion with 25 
mg diphenhydramine i.v., 650 mg acetaminophen p.o. and 50 mg hydrocortisone i.v. and 
will be closely monitored during the infusion and 4 hours after. All patients will be 
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Recombinant DNA Research, Volume 18 
